The long-term objective of this proposal is to elucidate novel roles played by ryanodine receptors (RyRs) in regulating sensory neuron function. We currently lack a sufficiently detailed understanding of how calcium signaling pathways regulate genetic changes that condition the inflammatory phenotype and influence neuronal survival. Current work suggests that calcium influx channels of the vanilloid receptor (VR) family are important components of inflammatory signaling pathways. Inflammatory signaling mediators such as bradykinin, ATP and nerve growth factor (NGF) regulate VR calcium influx channels, yet it is not known how these receptor systems couple to VR channel activation. Based on recent findings and our preliminary work, our hypothesis is that RyRs are integral regulators of VR or VR-like calcium channels as pivotal couplers linking calcium mobilizing receptors to activation of influx pathways. To begin to dissect the roles of RyRs in neuronal inflammatory responses, we propose to further develop the NG115-401L neuronal cell line as a model cell system for sensory neuron function. Thus, our first specific aim is to characterize the expression profiles of RyRs and calcium channels of the TRP family (including VR channels) in 401L cells and to perform co-immunoprecipitation studies to determine whether RyRs and TRP/TRP-like channels exist as physical complexes. In the second specific aim we take advantage of using the well-characterized homogeneous 401L cell line to produce cell lines deficient in RyR function. RyR deficient 401L cells represent a valuable tool to specifically assess the role of RyRs in calcium signaling pathways elicited by pro-inflammatory agents and the longer-term effects of impaired RyR function on gene expression and growth regulation, which constitutes the third specific aim of the project. A better understanding of RyR function in sensory neurons will greatly augment our ability to understand and rectify flawed RyR activity in inflammatory signaling pathways that lead to conditions of neuropathic pain and inflammation. [unreadable] [unreadable]